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Eft226, a First in Class Inhibitor of Eif4a, Targets FGFR1/2 and HER2

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eFT226, a first in class inhibitor of eIF4A, targets FGFR1/2 and HER2 driven cancers B133 Peggy A. Thompson, Nathan P. Young, Craig R. Stumpf, Boreth Eam, Vikas K. Goel, Joan Chen, Sarah Fish, Gregory S. Parker, Adina Gerson-Gurwitz, Maria Barrera, Eric Sung, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Kevin R. Webster, Siegfried H. Reich eFFECTOR Therapeutics, San Diego, CA Abstract Results Background: Mutations or amplifications affecting receptor tyrosine kinases (RTKs) activate eFT226 is a Sequence Selective Translational Regulator eFT226 Downregulates RTK Protein Expression and AKT/MAPK Signaling eFT226 Treatment Results in Tumor Growth Inhibition and Regression the RAS/MAPK and PI3K/AKT signaling pathways thereby promoting cancer cell MDA-MB-361 HER2 GAPDH proliferation and survival. Oncoprotein expression is tightly controlled at the level of mRNA A. B. A. A. MDA-MB-361 (BC: HER2amp) MDA-MB-361 (BC: HER2amp) translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) HO N 3’ Tumor Volume Body Weight complex consisting of eIF4A, eIF4E, and eIF4G. eIF4A functions to catalyze the unwinding of O HO eIF4A1 A/G 60S secondary structure in the 5’-untranslated region (5’-UTR) of mRNA facilitating ribosome 40S scanning and translation initiation. eFT226 is a first in class inhibitor that converts eIF4A1 N eFT226 mRNA 80S Polysomes into a sequence specific translational repressor. eFT226 increases the affinity between O O A/G 1 2 3 4 5 6 7 8 9 10 eIF4A1 and polypurine recognition elements in the 5’-UTR leading to selective Fraction 5’ downregulation of mRNA translation. The polypurine element is highly enriched in the 5’- CN B. C. UTR of eFT226 target genes, many of which are known oncogenic drivers, including MDA-MB-361 (HER2amp) NCI-H716 (FGFR2amp) FGFR1/2 and HER2, enabling eFT226 to selectively inhibit dysregulated oncogene C. 0 10 30 100 eFT226 (nM) 0 10 25 50 100 eFT226 (nM) expression. HER2 FGFR2 Binding to AGAGAG RNA surface RNA KD (µM) KD (µM) Fold Methods: 5’-UTR dependency was evaluated using cell-based luciferase reporter assays. Sequence w/ eFT226 w/o eFT226 Change p-ERK p-ERK Regulation of protein expression was analyzed by western blot analysis. Antitumor activity AGAGAG 0.021 ±0.001 8.0 ±0.9 381 ERK ERK B. NCI-H716 (CRC: FGFR2amp) C. NCI-H1581 (NSCLC: FGFR1amp) was assessed in vitro by proliferation and apoptosis assays. For in vivo experiments, GGCGGC 3.19 ±0.03 8.0 ±0.3 2.5 p-AKT S473 p-AKT S473 Tumor Volume Tumor Volume athymic nude or NOD/SCID mice were implanted with subcutaneous xenograft models of CCGCCG 9.6 ±0.4 3.27 ±0.06 0.34 AKT AKT FGFR1, FGFR2 or HER2 driven tumors and treated with eFT226 administered Q4D IV. CAACAA 2.43 ±0.01 3.78 ±0.05 1.6 GAPDH Tubulin Results: eFT226 inhibits the translation of FGFR1, FGFR2 and HER2 through formation of a sequence dependent ternary complex with eIF4A1 and polypurine elements within the 5’- UTR of mRNA [eIF4A1-eFT226-mRNA]. Formation of this ternary complex blocks ribosome D. MDA-MB-361 E. NCI-H716 scanning along the 5’-UTR leading to dose dependent inhibition of RTK protein expression. Cells transiently transfected with luciferase reporter constructs containing the 5’-UTR of D. 5’-UTR eFT226 Rel. to Hipp. Rel. to each RTK resulted in 10-45-fold greater sensitivity to inhibition by eFT226 compared to a Sequence IC50 (nM) AGAGAG IC50 (nM) AGAGAG control 5’-UTR confirming the 5’-UTR dependency. In solid tumor cell lines driven by AGAGAG 1.5 ±0.4 1 426 1 alterations in FGFR1, FGFR2 or HER2, downregulation of RTK expression by eFT226 resulted GGCGGC 13.8 ±2.0 9.2 204 0.5 in decreased MAPK and AKT signaling, potent inhibition of cell proliferation and an CCGCCG 92 ±18 61.5 369 0.9 induction of apoptosis suggesting that eFT226 could be effective in treating tumor types CAACAA 218 ±55 145 210 0.5 D. dependent on these oncogenic drivers. Solid tumor xenograft models harboring FGFR1/2 F. MDA-MB-361 G. NCI-H716 or HER2 amplifications treated with eFT226 resulted in significant in vivo tumor growth inhibition and regression at well tolerated doses in breast, non-small cell lung and colorectal cancer models. Treatment with eFT226 also decreased RTK protein levels supporting the potential to use these eFT226 target genes as pharmacodynamic markers of Figure 1. eFT226 is a sequence selective translational regulator. A) Chemical structure of eFT226. B) Schematic of the ternary complex interactions [eIF4A-eFT226-mRNA]. C) eFT226 enhances the binding target engagement. of eIF4A1 to specific RNAs. A representative Biacore sensogram of eIF4A1 binding to an AGAGAG RNA Conclusions: eFT226 is efficacious against tumor models with alterations in FGFR1, FGFR2 surface in the presence (green) or absence (black) of eFT226 (left panel). The stability of ternary and HER2 RTKs. The antitumor response observed in preclinical in vivo models driven by complexes formed on different RNA sequences is summarized (right panel). D) Inhibition of in vitro H. Proliferation Apoptosis Apoptosis RTK amplifications demonstrates the potential for eFT226 in the treatment of solid tumors translation by eFT226 is sequence dependent. Luciferase reporter gene constructs containing 5’-UTRs Cell line Tumor type RTK with FGFR1/2 or HER2 alterations. Furthermore, this data provides a means to select with 6-mer sequence motif repeats were transiently transfected into the MDA-MB-231 cell line and GI50 (nM) IC50 (nM) Emax sensitive patient subsets during clinical development. Clinical trials in patients with solid treated with increasing concentrations of eFT226 or hippuristanol for 4 hr. Green: AGAGAG, Blue: BT474 Breast HER2 4.4 2 3 right panel MDAMB361 Breast HER2 11.9 16 8 tumor malignancies have initiated. GGCGGC, Red: CCGCCG and Black: CAACAA. IC50 values are summarized in the table ( ). MDAMB231 Breast FGFR1 10.9 15 10 Figure 5. eFT226 inhibits the growth of RTK driven xenografts in vivo. A) MDA-MB-361 (HER2amp) MFM223 Breast FGFR2 3.1 6 14 xenograft-bearing animals were treated Q4D with either vehicle or eFT226 IV (1 mg/kg or 0.1 mg/kg) for NCIH716 Colorectal FGFR2 8.4 23 4 28 days. eFT226 treatment results in significant tumor regression (left panel). eFT226 is well tolerated as NCIH1581 Non-small cell lung FGFR1 5.7 90 1.6 Introduction Inhibition of eFT226 Target Genes is Mediated Through the 5’-UTR seen by the body weight measurements taken throughout the tumor growth study (right panel). B) amp Figure 3. eFT226 translationally regulates key oncogenic drivers resulting in inhibition of growth and Treatment of NCI-H716 (FGFR2 ) xenograft-bearing animals with the indicated doses of eFT226 Q4D RTKs A. amp survival. A) Polysome profile of MDA-MB-361 cells treated with 20 nM eFT226 or control for 3 hr (left results in tumor growth inhibition and regression after 15 days. C) Treatment of NCI-H1581 (FGFR1 ) 5’-UTR eFT226 panel). eFT226 inhibits the translation of HER2 (center panel) but not GAPDH (right panel) as shown by xenograft-bearing animals with the indicated doses of eFT226 Q4D results in significant tumor growth • eIF4A RNA helicase is an essential Sequence IC (nM) 50 qPCR analysis of mRNA from polysome fractions. B-C) Treatment of MDA-MB-361 (B) or NCI-H716 (C) cell inhibition after 20 days. D) Tumor growth inhibition values at day 14-15 in RTK (FGFR1, FGFR2 and HER2) component of the translation FGFR1 0.8 driven xenograft models for the indicated doses of eFT226 (Q4D IV). Dotted line indicates 100% tumor PI3K RAS lines with eFT226 for 24 hr decreases RTK protein levels, AKT and MAPK signaling as assessed by western initiation complex that regulates FGFR2 1.7 blot analysis. D-E) RTK and MAPK or AKT signaling are co-regulated. F-G) Down regulation of RTK protein growth inhibition. PTEN RAF multiple key oncogenes involved in HER2 4.2 levels results in inhibition of cell growth and an induction of apoptosis. H) Tabulated summary of AKT TUBA 36 proliferation (measured by Cell Titer-Glo) and apoptosis (measured by Caspase-Glo 3/7) values for RTK MEK tumor cell proliferation, survival and dependent cell lines treated with eFT226. metastasis Conclusions TSC ERK eFT226 Treatment Inhibits RTK Expression In vivo • RTK signaling activates pathways • eFT226 is a potent and selective inhibitor of eIF4A1 dependent B. C. amp that enhance eIF4A activity SNU-16 MDA-MB-361 A. NCI-H716 (FGFR2 ) B. translation mTOR wt FGFR2 (∆UTR) wt HER2 (∆UTR) • eFFECTOR Therapeutics has designed Vehicle eFT226 (1mg/kg) eFT226 (nM) eFT226 (nM) eFT226 (nM) eFT226 (nM) FGFR2 • eFT226 translationally down regulates oncogenic RTK protein 0 25 100 0 25 100 0 30 100 0 30 100 eFT226, a potent, small molecule p-Akt S473 expression (e.g., FGFR1/2 and HER2) through the formation of a S6K1 RSK FGFR2 HER2 sequence selective inhibitor of eIF4A Akt stable ternary complex [eIF4A-eFT226-mRNA] with specific 5’-UTR Cyclin D1 Cyclin D1 mediated translation Tubulin Degradation Tubulin Vinculin motifs P P C. NCI-H1581 (FGFR1amp) D. eIF4B PDCD4 • eFT226 inhibits tumor cell proliferation and induces apoptosis Figure 2. Protein expression of RTKs are regulated by eFT226 through the 5’-UTR. A) Luciferase reporter eFT226 gene constructs containing 5’-UTRs of FGFR1/2, HER2 or TUBA were transiently transfected into the Vehicle 0.3 mg/kg 1 mg/kg • eFT226 is well-tolerated and shows significant in vivo tumor growth HEK293t cells and treated with increasing concentrations of eFT226 for 4 hr. B-C) Predicted eFT226 FGFR1 inhibition and regression in RTK driven xenograft models binding sites were deleted from 5’-UTR (∆UTR) constructs.
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  • Inhibition of the MID1 Protein Complex

    Inhibition of the MID1 Protein Complex

    Matthes et al. Cell Death Discovery (2018) 4:4 DOI 10.1038/s41420-017-0003-8 Cell Death Discovery ARTICLE Open Access Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis Frank Matthes1,MoritzM.Hettich1, Judith Schilling1, Diana Flores-Dominguez1, Nelli Blank1, Thomas Wiglenda2, Alexander Buntru2,HannaWolf1, Stephanie Weber1,InaVorberg 1, Alina Dagane2, Gunnar Dittmar2,3,ErichWanker2, Dan Ehninger1 and Sybille Krauss1 Abstract Alzheimer’s disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-β (Aβ) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aβ peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aβ in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These 1234567890 1234567890 findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.